1. Field of Invention
The present invention relates generally to methods and systems for detecting a pathogen, and more particularly, to methods and systems using metal-enhanced fluorescence due to interactions of fluorophores with metallic particles and/or surfaces.
2. Description of the Related Art
The 2001 terror attacks in the United States clearly demonstrated a need for rapid detection systems for unequivocal identification of bio-warfare/bio-terrorism agents such as Bacillus Anthracis, the causative agent of anthrax. The ability to accurately identify biological threat agents in real time will enable first responders and clinicians to make informed decisions about the most appropriate countermeasures.
Bacillus anthracis, the virulent, endospore-forming bacterium notorious for its recent use as a bioterror weapon, has plagued humans and livestock for many years [34]. The bacterium was intimately associated with the founding of the sciences of bacteriology and immunology, highlighted by Pasteur's famous demonstration of vaccine protection of sheep at Pouilly-le-Fort, France in 1881 [35]. Since then, little attention has been focused on understanding the biology of the organism, save for the fact that it possesses properties that make it ideally suited as a biological weapon. It forms heat resistant spores that are easy to produce using commercially available technology and can infect via the aerosol route. It has been reported that at the time of the Gulf War, Iraq produced large quantities of anthrax spores and had deployed SCUD/Al-Hussein missiles equipped with biological weapons warheads [36].
Bacillus anthracis is the only obligate pathogen within the genus Bacillus, which comprises the Gram-positive aerobic or facultatively anaerobic spore-forming, rod-shaped bacteria. It is frequently convenient to class B. anthracis informally within the B. cereus group, which, on the basis of phenotype, comprises B. cereus, B. anthracis, B. thuringiensis and B. mycoides [37]. It is not possible to discriminate between species in this group based on 16S rRNA sequences. However, amplified fragment length polymorphism (AFLP) and multiple-locus VNTR (variable number tandem repeat) analysis (MLVA analysis) have provided clear evidence that B. anthracis can be distinguished reliably from other members of the bacilli [38,39]. In practical terms, the demonstration of virulence constitutes the principle point of difference between typical strains of B. anthracis and those of other anthrax-like organisms [40].
While Bacillus anthracis can kill a broad range of animals, other members of the Gram-positive Bacillus genus are typically soil-dwellers that cause, at worst, mild opportunistic infections. B. anthracis is a member of the B. cereus group of very closely-related, ubiquitous, soil bacteria [41,42] phylogenetically separate from the other completely sequenced Bacillus genomes, B. subtilis [41] and B. halodurans [43]. The nature of the virulence of individual strains in the B. cereus group is often determined by genes carried on large plasmids. For instance, B. thuringiensis strains are distinguished by their production of plasmid-encoded insecticidal crystal toxins (δ-endotoxins) of different specificity [44]. For B. anthracis, genes for the anthrax toxin complex and poly-D-glutamic acid capsule are found on plasmid pX01 (181.6 kb) [45] and pX02 (96 kb) [46] respectively. While the plasmid genes are necessary for full virulence, the contributions of chromosomal determinants to the complex pathogenesis of anthrax are still largely unknown.
The US anthrax bio-terrorism attack in the autumn of 2001 offered a tragic “proof of principle” of the danger of B. anthracis and has spurred numerous efforts by the biomedical community to improve forensics and medical countermeasures against the bacterium. In the key area of agent detection and warning, major advances have been made. For example, systems capable of detecting aerosolized agents have been developed and deployed based on DNA (BASIS) and antibody based technologies (Portal Shield). The ground breaking work of Department of Energy (DOE) researchers looking at aerosol samples from 11 major US cities found that nonpathogenic, close relatives of B. anthracis could be detected year round and that their abundance varied with the season [47]. However, experience gained from the field has shown that differentiating threat agents, particularly B. anthracis from other nonpathogenic dose relatives is challenging.
When compared to other bacterial genomes, most B. anthracis proteins have their highest level of similarity to other Bacillus genomes (B. subtilis (2065 (36%)) and B. halodurans (1082 (19%)). Most B. anthracis chromosomal proteins have homologs in the draft sequence of the B. cereus 10987 genome, confirming the very close relationship between these organisms. There are 642 genes in B. anthracis without matches in B. subtilis, B. halodurans or B. cereus 10987, but these are mostly small hypothetical proteins. Only 43 have a predicted function and these numbers may be lower when the completed B. cereus 10987 genome is available. These genes may define unique phenotypic characteristics of B. anthracis, which could be potentially of great interest in regard to virulence.
Traditional laboratory based methods have exploited these differences but can take several days to produce results. In the context of a biological attack this is an unacceptable delay if resulting casualties are to be minimized. The need for “real-time” (<60 mins) detection has lead to the development of technologies based on DNA (PCR) and protein (antibody) targets [37]. PCR and reverse transcriptase PCR assays have been reported for detecting anthrax [1,2] in air samples [5], anthrax spore detection by flow cytometry [6], microsonication to disrupt bacterial spores [7] and real-time devices for PCR analysis [8]. However, these advances are not considered simple or monetarily reasonable, and therefore limit their potential as field-deployable, emerging technologies for use in ultra-sensitive pathogen detection.
Other methods include use of a detection label such as a linked fluorescent dye molecule, such as fluorescein isothiocyanate, rhodamine, Cascade blue, that absorb electromagnetic energy in a particular absorption wavelength spectrum and subsequently emit visible light at one or more longer (i.e., less energetic) wavelengths. The fluorescent molecules (fluorophores) can be detected by illumination with light of an appropriate excitation frequency and the resultant spectral emissions can be detected by electro-optical sensors or light microscopy. A wide variety of fluorescent dyes are available and offer a selection of excitation and emission spectra. Unfortunately, detection methods that employ fluorescent labels are of limited sensitivity for a variety of reasons. Firstly, the lifetime of the fluorescence emission is usually short, on the order of 1 to 100 ns. Further, the limit of detection of from typical fluorophores is limited by the significant background noise contributed by nonspecific fluorescence and reflected excitation light. Additionally, organic dye fluorophores are susceptible to photolytic decomposition of the dye molecule (i.e., photobleaching). Thus, even in situations where background noise is relatively low, it is often not possible to integrate a weak fluorescent signal over a long detection time, since the dye molecules decompose as a function of incident irradiation in the UV and near-UV bands.
Thus, there is a need for a detection method and system using fluorophores that identifies the pathogenic agent and preferably able to differentiate between multiple agents, that does not suffer from the problems of the prior art and does not require any amplification steps, such as in PCR or ELISA.